U.S. patent application number 15/278841 was filed with the patent office on 2017-04-06 for transgenic nuclease systems and methods.
The applicant listed for this patent is Agenovir Corporation. Invention is credited to Stephen R. Quake.
Application Number | 20170096649 15/278841 |
Document ID | / |
Family ID | 58427863 |
Filed Date | 2017-04-06 |
United States Patent
Application |
20170096649 |
Kind Code |
A1 |
Quake; Stephen R. |
April 6, 2017 |
TRANSGENIC NUCLEASE SYSTEMS AND METHODS
Abstract
The invention provides transgenic organisms that include a
transgene that codes for a product that can be used to digest
foreign nucleic acid. The transgene can code for a targeting
nuclease, a guide sequence, or other components of a guided
nuclease system. Expression of the transgene causes the organism to
express an active targeting nuclease that targets and digests
foreign nucleic acid. The targeting nuclease targets the foreign
nucleic acid specifically and avoids targeting the organism's
native genetic material.
Inventors: |
Quake; Stephen R.;
(Stanford, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agenovir Corporation |
South San Francisco |
CA |
US |
|
|
Family ID: |
58427863 |
Appl. No.: |
15/278841 |
Filed: |
September 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62236271 |
Oct 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 301/00 20130101;
A01K 67/0275 20130101; A01K 2217/206 20130101; C12N 15/8222
20130101; A01K 2267/02 20130101; A01K 2217/052 20130101; C12N 9/22
20130101 |
International
Class: |
C12N 9/22 20060101
C12N009/22; A01K 67/027 20060101 A01K067/027; C12N 15/82 20060101
C12N015/82 |
Claims
1. A non-human transgenic organism comprising a transgene, wherein
the transgene comprises nucleic acid that encodes a targeting
nuclease that can be activated to digest foreign nucleic acid.
2. The organism of claim 1, further comprising a feature that
promotes expression of the transgene.
3. The organism of claim 2, wherein the feature that promotes
expression comprises a promoter that selectively favors expression
of the targeting nuclease within a certain tissue or cell type of
the organism.
4. The composition of claim 2, wherein the nuclease is one selected
from the group consisting of a zinc-finger nuclease, a
transcription activator-like effector nuclease, and a
meganuclease.
5. The organism of the claim 1, wherein the organism is a plant
crop or mammalian livestock, and further wherein the targeting
nuclease uses a targeting sequence to target and digest the foreign
nucleic acid without digesting a genome of the organism.
6. The organism of claim 5, wherein the nuclease comprises Cas9
endonuclease and the targeting sequence comprises a guide RNA.
7. The organism of claim 6, wherein the guide RNA has no match
according to predetermined similarity criteria within the
genome.
8. The organism of claim 7, wherein the predetermined similarity
criteria require that the guide RNA has no match >60% within the
genome.
9. The organism of claim 5, wherein the targeting sequence is
encoded adjacent the targeting nuclease within a complex in the
transgene, and the complex is transcribed together as a single
primary transcript.
10. The organism of claim 9, wherein activation of the targeting
nuclease includes causing the complex to be transcribed.
11. The organism of claim 1, wherein activating the targeting
nuclease includes administering an agent to cause expression of the
transgene.
12. The organism of claim 1, wherein activating the targeting
nuclease includes causing expression of the targeting nuclease from
the transgene and causing the targeting nuclease to digest viral
foreign nucleic acid.
13. The organism of claim 1, wherein the organism is a plant.
14. The organism of claim 1, wherein the organism is an animal.
15. A method of making a non-human transgenic organism, the method
comprising: introducing into a cell a transgene encoding a
targeting nuclease; integrating the transgene into heritable
genetic material of the cell; and growing the cell into an organism
for agricultural use, wherein cells of the organism include the
transgene.
16. The method of claim 15, wherein the organism is a mammal and
the cell comprises an oocyte or a cell of an embryo and wherein
growing the cell into the organism includes transfer of the oocyte
or embryo into a recipient female.
17. The method of claim 16, wherein the targeting nuclease
comprises Cas9 endonuclease under control of a promoter.
18. The method of claim 15, wherein the organism is a plant and the
targeting nuclease comprises Cas9 endonuclease.
19. The method of claim 15, wherein the organism is a plant crop or
mammalian livestock and the targeting nuclease comprises Cas9
endonuclease.
20. The method of claim 19, wherein the transgene also encodes at
least one guide sequence that, when transcribed into a guide RNA,
guides the Cas9 endonuclease to digest nucleic acid foreign to the
organism.
Description
RELATED APPLICATION
[0001] This application claims the benefit of and priority to U.S.
Provisional No. 62/236,271, filed Oct. 2, 2015, which is
incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates to transgenic agricultural
organisms.
BACKGROUND
[0003] Infections are a significant problem in agriculture. Virus
epidemics of plants and livestock have increased steadily since the
Neolithic period. As humans became dependent on agriculture and
farming, diseases such as potyvirus of potatoes and rinderpest of
cattle had devastating consequences. One of the first viruses to be
discovered, tobacco mosaic virus, has cost billions of dollars in
crop losses. Another important pathogen of animals and plants are
the viruses of the family Rhabdoviridae, which includes rabies.
Another example is a virus from family Potyviridae known as the
Tulip breaking virus (TBV). TBV has dramatic effects on the colors
of tulip petals, which in the Netherlands in the 17th century
contributed to the skyrocketing prices of rare tulip bulbs, but
which also retards the ability of the tulip to propagate.
[0004] Viruses of livestock are myriad. For example, different
species of the Alphaviruses variously infect horses and farmed
fish. The Pestiviruses are associated with swine fever and bovine
viral diarrhea/Mucosal disease (BVD/MD). The Arteriviridae family
includes equine arteritis virus (EAV) and porcine reproductive and
respiratory syndrome virus (PRRSV). Other viruses or families of
viruses with dramatic effects on agriculture include coronaviruses,
paramyxoviruses, Hendra and Nipah virus, avian influenza,
Bluetongue virus (BTV), Porcine Circoviruses (PCV), and African
swine fever. There is quite a wide variety of viruses that have a
devastating financial effect on agricultural and that do
significant harm to animal welfare.
SUMMARY
[0005] The invention provides transgenic organisms that include a
transgene that codes for a product that can be used to digest
foreign nucleic acid. The transgene can code for a targeting
nuclease, a guide sequence, or other components of a guided
nuclease system. The living organism expresses the transgene
itself, allowing it to express an active targeting nuclease that
targets and digests foreign nucleic acid. The nuclease is
preferably a programmable nuclease. The nuclease can be, for
example, a zinc finger nuclease, a meganuclease, a TALENs, Cpf1,
PfAgo, or NgAgo, and is preferably Cas9. The targeting nuclease
targets the foreign nucleic acid specifically and avoids targeting
the organism's native genetic material. For example, the transgene
may encode a Cas9 enzyme that, when expressed, uses a guide RNA
sequence complementary to the foreign nucleic acid to bind to, and
make cuts in, that foreign nucleic acid. The guide sequence may be
encoded by the transgene in the organism in the first instance or
may be integrated into a CRISPR/Cas9 complex that includes the
transgene within the organism's somatic cells in response to
infection by the operation of the CRISPR/Cas9 machinery. Where the
guide sequence is complementary to a target within viral nucleic
acid and has no corresponding complementary portion within the
organism's genome, the targeting nuclease may digest viral genetic
material, thereby protecting the organism from the adverse effects
of a viral infection.
[0006] Transgene expression may be constitutive or may be
controlled by a suitable control mechanism, such as a
tissue-specific promoter or controlled by an exogenous agent such
as a small molecule. While discussed above in terms of digesting
viral nucleic acid, the transgene may be activated or expressed to
digest or cut any foreign nucleic acid including, for example, from
bacteria or other parasites. Additionally or alternatively, the
transgene product may in fact target features of the organism's
genome, e.g., to initiate expression or repression of some gene
product at some point in the organism's life. Since the transgene
can specifically digest foreign nucleic acid such as viral genetic
material without interfering with the organism's genome, the
organism can have innate protection from the adverse effects of an
infection. Since the transgenic organism carries innate protection
against infection, the use of the organism in agriculture can avoid
the devastating financial impacts and the harms to animal welfare
that have characterized agriculture for millennia.
[0007] In certain aspects, the invention provides a method of
making a non-human transgenic organism. The method includes
introducing into a cell a transgene encoding a targeting nuclease,
integrating the transgene into heritable genetic material of the
cell, and growing the cell into an organism for agricultural use,
wherein cells of the organism include the transgene. In some
embodiments, the organism is a mammal for livestock and the cell
comprises an oocyte or a cell of an embryo and wherein growing the
cell into the organism includes transfer of the oocyte or embryo
into a recipient female. The targeting nuclease may include Cas9
endonuclease under control of a promoter. In certain embodiments,
the organism is a plant and the targeting nuclease comprises Cas9
endonuclease. Thus preferably the organism is a plant crop or
mammalian livestock and the targeting nuclease includes Cas9
endonuclease.
[0008] The transgene may also encode at least one guide sequence
that, when transcribed into a guide RNA, guides the Cas9
endonuclease to digest nucleic acid foreign to the organism.
Preferably, the guide RNA has no match according to a predetermined
similarity criteria within the genome. For example, the similarity
criteria may require that the guide RNA has no match >60% within
the genome.
[0009] Aspects of the invention provide a non-human transgenic
organism comprising a transgene. The transgene includes nucleic
acid that encodes a targeting nuclease that can be activated to
digest foreign nucleic acid. The organism may include a feature
that promotes expression of the transgene. For example, the feature
that promotes expression may be a promoter-enhancer cassette that
selectively favors expression of the targeting nuclease within a
certain tissue or cell type of the organism. In some embodiments,
the transgene is under the control of an inducible promoter.
Suitable inducible promoters include, for example, the tetracycline
on system or the tetracycline off system. In certain embodiments,
the nuclease is expressed constitutively. In some embodiments, the
nuclease is one selected from the group consisting of a zinc-finger
nuclease, a transcription activator-like effector nuclease, and a
meganuclease. In preferred embodiments, the nuclease comprises Cas9
endonuclease and the targeting sequence comprises a guide RNA. The
organism may be a plant crop or mammalian livestock, and the
targeting nuclease may use a targeting sequence to target and
digest the foreign nucleic acid without digesting a genome of the
organism.
[0010] In some embodiments, the guide RNA has no match according to
a predetermined similarity criteria within the genome of the
transgenic organism (e.g., the guide RNA has no match >60%
within the genome). In certain embodiments, the targeting sequence
is encoded adjacent the targeting nuclease within a complex in the
transgene, and the complex is transcribed together as a single
primary transcript. Activation of the targeting nuclease includes
causing the complex to be transcribed. The nuclease may be
activated by administration of an agent such as a small molecule.
In some embodiments, activating the targeting nuclease includes
causing expression of the targeting nuclease from the transgene and
causing the targeting nuclease to digest viral foreign nucleic
acid.
[0011] In some embodiments, the organism is a plant such as corn,
wheat, maize, rapeseed, soybean, sunflower, barley, sorghum,
potato, or rice. In certain embodiments, the organism is an animal
(e.g., cattle, horse, goat, sheep, swine, and poultry).
[0012] Aspects of the invention provide a seed for a crop plant.
The seed includes at least one transgene in its heritable genetic
material, in which the transgene encodes a targeting nuclease. The
targeting nuclease may be a Cas9 endonuclease. The crop plant may
be, for example, corn, wheat, maize, rapeseed, soybean, sunflower,
barley, sorghum, potato, and rice. In some embodiments, the
transgene also encodes at least one guide sequence that, when
transcribed into a guide RNA, guides the Cas9 endonuclease to
digest nucleic acid foreign to the crop plant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 diagrams a method for making a non-human transgenic
organism.
[0014] FIG. 2 shows a composition for introducing a transgene into
a cell.
[0015] FIG. 3 diagrams a vector according to certain
embodiments.
[0016] FIG. 4 gives results of digesting foreign nucleic acid.
[0017] FIG. 5 shows use of zinc-finger targeting nuclease.
[0018] FIG. 6 describes an exemplary method for selecting a
gRNA.
[0019] FIG. 7 outlines a similarity criteria according to certain
embodiments.
[0020] FIG. 8 diagrams the avian flu virus genome for targets for
cleavage.
[0021] FIG. 9 shows a genome of a Bluetongue virus (BTV) for
targeting.
[0022] FIG. 10 diagrams the tobacco mosaic virus genetic
material.
[0023] FIG. 11 shows parts of the genome of banana bunchy top virus
(BBTV).
[0024] FIG. 12 shows a gel resulting from a CRISPR assay.
[0025] FIG. 13 shows a composition that includes an EGFP marker
fused after the Cas9 protein.
[0026] FIG. 14 shows gRNA targets along a reference genome.
[0027] FIG. 15 gives genome context around guide RNA sgEBV3/4/5 and
PCR primer locations.
[0028] FIG. 16 shows large deletions induced by sgEBV3/5 and
sgEBV4/5.
[0029] FIG. 17 shows that Sanger sequencing confirmed genome
cleavage and repair ligation.
DETAILED DESCRIPTION
[0030] Aspects of the invention relate to agricultural/biological
(agbio) applications of targetable nucleases and particularly to
transgenic organisms such as plants or animals. In some
embodiments, the invention provides an organism such as an animal
or plant, or a seed for a plant, that itself expresses a targeting
nuclease. Additionally, the invention provides methods of creating
a transgenic organism that expresses a targeting nuclease. In some
embodiments, the organism uses the targeting nuclease to cleave
foreign nucleic acid. Additionally or alternatively, a transgenic
organism of the invention can use a targeting nuclease to affect
gene expression (e.g., by interfering with a promotor or
effectively performing a knock-out or knock-in via the transgene).
The transgenic organism can express a targeting nuclease, such as
Cas9, either in every cell or in tissue specific ways. It can also
be expressed constitutively or conditionally, e.g., externally
inducible by small molecule activation.
[0031] FIG. 1 diagrams a method of making a non-human transgenic
organism. The ability to introduce genes and/or other DNA sequences
into the germline or somatic cells of organisms such as mammalian
livestock or plants results in germline changes that are inherited
by the offspring of the animals and all cells of these offspring
inherit the introduced transgene. In the depicted method, a cell
such as an oocyte or a cell within an embryo is addressed. A
transgene is obtained to be integrated into the cell. The transgene
may include a gene for a targeting nuclease and may optionally
further include a targeting sequence. The transgene is introduced
into the cell and the organism is grown to create the transgenic
organism. In the preferred embodiment, the transgenic organism may
express the transgene to cleave foreign nucleic acid.
Transgenic Organism
[0032] The production of transgenic animals is commonly done in
various ways. Transgenic organisms may be produced by targeted
insertion of DNA by homologous recombination in embryonic stem (ES)
cells or by pronuclear injection of a fertilized ovum and
integration of DNA. It is also possible to introduce a transgene
via vector such as a plasmid or virus. For example, retroviral
vector systems are based on lentiviruses, a small subgroup of the
retroviruses.
[0033] Several methods for introducing foreign DNA into the
germline of mammals have been developed. The techniques allow the
mixing of cells from different embryos, i.e. chimera production,
introducing pluripotent cells such as ES cells into developing
embryos, micro-injecting DNA, and infection by retroviruses. Some
techniques include removing fertilized eggs or early embryos,
culturing them in vitro and then returning them to recipient
mothers where further embryogenesis can proceed. In other
embodiments, a lentiviral vector can be introduced throughout the
development of the organism. Thus in one embodiment the cell is a
perinatal cell, which could be an embryonic cell (e.g., in utero).
Preferably the cell is an oocyte, an oviduct cell, an ovarian cell,
an ovum, an ES cell, a blastocyte, a spermatocyte, a spermatid, a
spermatozoa, or a spermatogonia.
[0034] It is possible to insert a foreign gene into mammalian
livestock embryos or plant germline cells, and for these genes to
be incorporated into the genome of the resulting animal. Insertion
of the foreign genes has been carried out mechanically (by
microinjection), and with the aid of retrovirus vectors. See e.g.,
Huszar et al., 1985, Insertion of a bacterial gene into the mouse
germ line using an infectious retrovirus vector PNAS 82:8587-8591,
incorporated by reference.
[0035] The introduced transgene can be sexually transmitted through
subsequent generations and are frequently expressed in the animal.
In some instances the proteins encoded by the foreign genes are
expressed in specific tissues. For example, the metallothionein
promoter has been used to direct the expression of the rat growth
hormone gene in the liver tissue of transgenic mice (Palmiter et
al., 1982, Dramatic growth of mice that develop from eggs
microinjected with metallothionein-growth hormone fusion genes,
Nature 300:611-615). Another example is the elastase promoter,
which has been shown to direct the expression of foreign genes in
the pancreas (Ornitz et al., 1985 Nature 313:600). Developmental
control of gene expression has also been achieved in transgenic
animals, i.e., the foreign gene is transcribed only during a
certain time period, and only in a particular tissue. For example,
Magram et al. (1985, Nature 315:338) demonstrated developmental
control of genes under the direction of a globin promoter; and
Krumlauf et al. (1985, Mol. Cell. Biol. 5:1639) demonstrated
similar results using the alpha-feto protein mini-gene.
[0036] The described methods can be used to generate transgenic,
non-human plants or animals or site specific gene modifications in
cell lines. Transgenic cells include one or more nucleic acids
according to the subject invention present as a transgene, where
included within this definition are the parent cells transformed to
include the transgene and the progeny thereof.
[0037] A transgenic animal may be made starting with stem cells. An
ES cell line may be employed, or embryonic cells may be obtained
freshly from a host, e.g. cow, pig, chicken, etc. Such cells are
grown on an appropriate fibroblast-feeder layer or grown in the
presence of leukemia inhibiting factor (LIF). When ES or embryonic
cells are transformed (e.g., using a vector of FIG. 2 or FIG. 3),
they may be used to produce transgenic animals. After
transformation, the cells are plated onto a feeder layer in an
appropriate medium. Cells containing the construct may be detected
by employing a selective medium. After sufficient time for colonies
to grow, they are picked and analyzed for the occurrence of
homologous recombination or integration of the construct. Those
colonies that are positive may then be used for embryo manipulation
and blastocyst injection. Blastocysts are obtained from 4 to 6 week
old super-ovulated females. The ES cells are trypsinized, and the
modified cells are injected into the blastocoel of the blastocyst.
After injection, the blastocysts are returned to each uterine horn
of pseudo-pregnant females. Females are then allowed to go to term
and the resulting offspring screened for the construct. By
providing for a different phenotype of the blastocyst and the
genetically modified cells, chimeric progeny can be readily
detected. The chimeric animals are screened for the presence of the
modified gene and males and females having the modification may be
mated to produce homozygous progeny. The transgenic animals may be
any non-human livestock mammal or any other suitable animal.
[0038] Transgenic plants may be produced in a similar manner.
Methods of preparing transgenic plant cells and plants are
described in U.S. Pat. Nos. 5,767,367; 5,750,870; 5,739,409;
5,689,049; 5,689,045; 5,674,731; 5,656,466; 5,633,155; 5,629,470;
5,595,896; 5,576,198; 5,538,879; 5,484,956; the disclosures of
which are herein incorporated by reference. Methods of producing
transgenic plants are also reviewed in Plant Biochemistry and
Molecular Biology (Eds. Lea & Leegood, John Wiley &
Sons)(1993) pp 275-295. In brief, a suitable plant cell or tissue
is harvested, depending on the nature of the plant species. As
such, in certain instances, protoplasts will be isolated, where
such protoplasts may be isolated from a variety of different plant
tissues, e.g. leaf, hypoctyl, root, etc. For protoplast isolation,
the harvested cells are incubated in the presence of cellulases in
order to remove the cell wall, where the exact incubation
conditions vary depending on the type of plant and/or tissue from
which the cell is derived. The resultant protoplasts are then
separated from the resultant cellular debris by sieving and
centrifugation. Instead of using protoplasts, embryogenic explants
comprising somatic cells may be used for preparation of the
transgenic host. Following cell or tissue harvesting, exogenous DNA
of interest is introduced into the plant cells, where a variety of
different techniques are available for such introduction. With
isolated protoplasts, the opportunity arise for introduction via
DNA-mediated gene transfer protocols, including: incubation of the
protoplasts with naked DNA, e.g. plasmids, comprising the exogenous
coding sequence of interest in the presence of polyvalent cations,
e.g. PEG or PLO; and electroporation of the protoplasts in the
presence of naked DNA comprising the exogenous sequence of
interest. Protoplasts that have successfully taken up the exogenous
DNA are then selected, grown into a callus, and ultimately into a
transgenic plant through contact with the appropriate amounts and
ratios of stimulatory factors, e.g. auxins and cytokinins. With
embryogenic explants, a convenient method of introducing the
exogenous DNA in the target somatic cells is through the use of
particle acceleration or "gene-gun" protocols. The resultant
explants are then allowed to grow into chimera plants, cross-bred
and transgenic progeny are obtained. Instead of the naked DNA
approaches described above, another convenient method of producing
transgenic plants is Agrobacterium mediated transformation. With
Agrobacterium mediated transformation, co-integrative or binary
vectors comprising the exogenous DNA are prepared and then
introduced into an appropriate Agrobacterium strain, e.g. A.
tumefaciens. The resultant bacteria are then incubated with
prepared protoplasts or tissue explants, e.g. leaf disks, and a
callus is produced. The callus is then grown under selective
conditions, selected and subjected to growth media to induce root
and shoot growth to ultimately produce a transgenic plant. Although
in general the techniques mentioned herein are well known in the
art, reference may be made in particular to Sambrook et al.,
Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley
& Sons, Inc.
Vectors for Introducing Transgene Into Cell
[0039] Methods of the invention may include using a vector to
introduce a transgene into a cell such as an oocyte or a cell
within an embryo.
[0040] FIG. 2 shows a composition for introducing a transgene into
a cell. The composition preferably includes a DNA strand (circular
or linear, here shown as circularized) that includes at least
nuclease gene and at least one targeting sequence (labelled gRNA in
FIG. 2). The composition may include an origin of replication such
as an HPV origin. Preferably, the composition includes one or more
promoters, any or all of which may be specific to keratinocytes.
Any suitable promoter or enhancer may be used that results in
expression within keratinocytes. For example, a nuclease may be
provided within a vector (e.g., a plasmid) that includes one or
more inducible promoters such as metallothionein (MT) and
1,24-vitamin D(3)(OH)(2) dehydroxylase (VDH) promoters responded to
the inducing agents, Cadmium and 1,25-vitamin D(3)(OH)(2)
(VitD(3)), respectively. In one embodiment, the nuclease is
Cas9.
[0041] FIG. 3 diagrams a vector according to certain embodiments.
The vector shown in FIG. 3 may be transfected into an oocyte or a
germ cell that is then matured into an agricultural organism. When
the organism grows, it will express active Cas9, which may then
digest foreign nucleic acid.
[0042] FIG. 4 gives results of digesting foreign nucleic acid. The
nuclease forms a complex with the gRNA (e.g., crRNA+tracrRNA or
sgRNA). The complex cuts the viral nucleic acid in a targeted
fashion to incapacitate the viral genome. The Cas9 endonuclease
causes a double strand break in the viral genome. By targeted
several locations along the viral genome and causing not a single
strand break, but a double strand break, the genome is effectively
cut a several locations along the genome. In a preferred
embodiment, the double strand breaks are designed so that small
deletions are caused, or small fragments are removed from the
genome so that even if natural repair mechanisms join the genome
together, the genome is render incapacitated.
[0043] A transgenic organism (e.g., crop plant or livestock mammal)
may be produced using a non-primate lentiviral expression vector.
Some vectors used in recombinant DNA techniques allow entities,
such as a segment of DNA (such as a heterologous DNA segment, such
as a heterologous cDNA segment), to be transferred into a host cell
for the purpose of replicating the vectors comprising a segment of
DNA. Examples of vectors used in recombinant DNA techniques include
but are not limited to plasmids, chromosomes, artificial
chromosomes or viruses. In a typical vector for use in the method
of the present invention, at least part of one or more protein
coding regions essential for replication may be removed from the
virus. This makes the viral vector replication-defective. Portions
of the viral genome may also be replaced by a library encoding
e.g., a targeting nuclease operably linked to a regulatory control
region and a reporter moiety in the vector genome in order to
generate a vector comprising candidate transgenes which is capable
of transducing a target cell and/or integrating its genome into the
genome. Lentivirus vectors are part of a larger group of retroviral
vectors. A detailed list of lentiviruses may be found in Coffin et
al ("Retroviruses" 1997 Cold Spring Harbor Laboratory Press Eds: J
M Coffin, S M Hughes, H E Varmus pp 758-763). In brief,
lentiviruses can be divided into primate and non-primate groups.
Examples of primate lentiviruses include but are not limited to:
the human immunodeficiency virus (HIV) and the simian
immunodeficiency virus (SIV). The non-primate lentiviral group
includes the prototype "slow virus" visna/maedi virus (VMV), as
well as the related caprine arthritis-encephalitis virus (CAEV),
equine infectious anaemia virus (EIAV) and the more recently
described feline immunodeficiency virus (FIV) and bovine
immunodeficiency virus (BIV). A distinction between the lentivirus
family and other types of retroviruses is that lentiviruses have
the capability to infect both dividing and non-dividing cells
(Lewis et al 1992 EMBO. J 11: 3053-3058; Lewis and Emerman 1994 J.
Virol. 68: 510-516). In contrast, other retroviruses--such as
MLV--are unable to infect non-dividing or slowly dividing cells
such as those that make up, for example, muscle, brain, lung and
liver tissue.
[0044] A "non-primate" vector, as used herein in some aspects of
the present invention, refers to a vector derived from a virus
which does not primarily infect primates, especially humans. Thus,
non-primate virus vectors include vectors which infect non-primate
mammals, such as dogs, sheep and horses, reptiles, birds and
insects. The non-primate lentivirus may be any member of the family
of lentiviridae which does not naturally infect a primate and may
include a feline immunodeficiency virus (FIV), a bovine
immunodeficiency virus (BIV), a caprine arthritis encephalitis
virus (CAEV), a Maedi visna virus (MVV) or an equine infectious
anaemia virus (EIAV). Preferably the lentivirus is an EIAV. Equine
infectious anaemia virus infects all equidae resulting in plasma
viremia and thrombocytopenia (Clabough, et al. 1991. J Virol.
65:6242-51). Virus replication is thought to be controlled by
maturation of monocytes into macrophages.
[0045] In one embodiment the viral vector is derived from EIAV.
EIAV has the simplest genomic structure of the lentiviruses and is
particularly preferred for use in the present invention. In
addition to the gag, pol and env genes EIAV encodes three other
genes: tat, rev, and S2. Tat acts as a transcriptional activator of
the viral LTR (Derse and Newbold 1993 Virology. 194:530-6; Maury,
et al 1994 Virology. 200:632-42) and Rev regulates and coordinates
the expression of viral genes through rev-response elements (RRE)
(Martarano et al 1994 J Virol. 68:3102-11). The mechanisms of
action of these two proteins are thought to be broadly similar to
the analogous mechanisms in the primate viruses (Martano et al
ibid). The function of S2 is unknown. In addition, an EIAV protein,
Ttm, has been identified that is encoded by the first exon of that
spliced to the env coding sequence at the start of the
transmembrane protein.
[0046] The viral RNA of this aspect of the invention is transcribed
from a promoter, which may be of viral or non-viral origin, but
which is capable of directing expression in a eukaryotic cell such
as a mammalian cell. Optionally an enhancer is added, either
upstream of the promoter or downstream. The RNA transcript is
terminated at a polyadenylation site which may be the one provided
in the lentiviral 3' LTR or a different polyadenylation signal.
[0047] A DNA transcription unit comprising a promoter and
optionally an enhancer capable of directing expression of a
non-primate lentiviral vector genome may be used. Transcription
units as described herein comprise regions of nucleic acid
containing sequences capable of being transcribed. The sequences
may be in the sense or antisense orientation with respect to the
promoter. Antisense constructs can be used to inhibit the
expression of a gene in a cell according to well-known techniques.
Nucleic acids may be, for example, ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or analogues thereof. Sequences
encoding mRNA will optionally include some or all of 5' and/or 3'
transcribed but untranslated flanking sequences naturally, or
otherwise, associated with the translated coding sequence. It may
optionally further include the associated transcriptional control
sequences normally associated with the transcribed sequences, for
example transcriptional stop signals, polyadenylation sites and
downstream enhancer elements. Nucleic acids may comprise cDNA or
genomic DNA (which may contain introns).
[0048] The basic structure of a retrovirus genome is a 5' LTR and a
3' LTR, between or within which are located a packaging signal to
enable the genome to be packaged, a primer binding site,
integration sites to enable integration into a host cell genome and
gag, pol and env genes encoding the packaging components--these are
polypeptides required for the assembly of viral particles. More
complex retroviruses have additional features, such as rev and RRE
sequences in HIV, which enable the efficient export of RNA
transcripts of the integrated provirus from the nucleus to the
cytoplasm of an infected target cell.
[0049] In the provirus, these genes are flanked at both ends by
regions called long terminal repeats (LTRs). The LTRs are
responsible for proviral integration, and transcription. LTRs also
serve as enhancer-promoter sequences and can control the expression
of the viral genes. Encapsidation of the retroviral RNAs occurs by
virtue of a psi sequence.
[0050] The LTRs themselves are identical sequences that can be
divided into three elements, which are called U3, R and U5. U3 is
derived from the sequence unique to the 3' end of the RNA. R is
derived from a sequence repeated at both ends of the RNA and U5 is
derived from the sequence unique to the 5' end of the RNA. The
sizes of the three elements can vary considerably among different
retroviruses. In a defective retroviral vector genome gag, pol and
env may be absent or not functional. The R regions at both ends of
the RNA are repeated sequences. U5 and U3 represent unique
sequences at the 5' and 3' ends of the RNA genome respectively.
[0051] The retroviral vector employed in the aspects of the present
invention may be derived from or may be derivable from any suitable
retrovirus. A large number of different retroviruses have been
identified. Examples include: murine leukemia virus (MLV), human
immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV),
mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV),
Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus
(Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine
sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV),
Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis
virus (AEV). A detailed list of retroviruses may be found in Coffin
et al., 1997, "retroviruses", Cold Spring Harbour Laboratory Press
Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763.
Targetable Nuclease
[0052] Methods of the invention include creating a transgenic
organism that expresses a targeting nuclease. Any suitable
targeting nuclease can be used including, for example, zinc-finger
nucleases (ZFNs), transcription activator-like effector nucleases
(TALENs), clustered regularly interspaced short palindromic repeat
(CRISPR) nucleases, meganucleases, other endo- or exo-nucleases, or
combinations thereof. See Schiffer, 2012, Targeted DNA mutagenesis
for the cure of chronic viral infections, J Virol 88(17):8920-8936,
incorporated by reference. In certain embodiments, the targeting
nuclease may be a DNA-guided nuclease (e.g., a Pyrococcus furiosus
Argonaute (PfAgo) or Natronobacterium gregoryi Argonaute (NgAgo).
The targeting nuclease may be a high-fidelity Cas9 (hi-fi Cas9),
e.g., as described in Kleinstiver et al., 2016, High-fidelity
CRISPR-Cas9 nucleases with no detectable genome-wide off-target
effects, Nature 529:490-495, incorporated by reference.
[0053] CRISPR methodologies employ a nuclease, CRISPR-associated
(Cas9), that complexes with small RNAs as guides (gRNAs) to cleave
DNA in a sequence-specific manner upstream of the protospacer
adjacent motif (PAM) in any genomic location. CRISPR may use
separate guide RNAs known as the crRNA and tracrRNA. These two
separate RNAs have been combined into a single RNA to enable
site-specific mammalian genome cutting through the design of a
short guide RNA. Cas9 and guide RNA (gRNA) may be synthesized by
known methods. Cas9/guide-RNA (gRNA) uses a non-specific DNA
cleavage protein Cas9, and an RNA oligo to hybridize to target and
recruit the Cas9/gRNA complex. See Chang et al., 2013, Genome
editing with RNA-guided Cas9 nuclease in zebrafish embryos, Cell
Res 23:465-472; Hwang et al., 2013, Efficient genome editing in
zebrafish using a CRISPR-Cas system, Nat. Biotechnol 31:227-229;
Xiao et al., 2013, Chromosomal deletions and inversions mediated by
TALENS and CRISPR/Cas in zebrafish, Nucl Acids Res 1-11, each
incorporated by reference.
[0054] CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) is found in bacteria and is believed to protect the
bacteria from phage infection. It has recently been used as a means
to alter gene expression in eukaryotic DNA, but has not been
proposed as an anti-viral therapy or more broadly as a way to
disrupt genomic material. Rather, it has been used to introduce
insertions or deletions as a way of increasing or decreasing
transcription in the DNA of a targeted cell or population of cells.
See for example, Horvath et al., Science (2010) 327:167-170; Terns
et al., Current Opinion in Microbiology (2011) 14:321-327; Bhaya et
al. Annu Rev Genet (2011) 45:273-297; Wiedenheft et al. Nature
(2012) 482:331-338); Jinek Met al. Science (2012) 337:816-821; Cong
L et al. Science (2013) 339:819-823; Jinek M et al. (2013) eLife
2:e00471; Mali P et al. (2013) Science 339:823-826; Qi L S et al.
(2013) Cell 152:1173-1183; Gilbert L A et al. (2013) Cell
154:442-451; Yang H et al. (2013) Cell 154:1370-1379; and Wang H et
al. (2013) Cell 153:910-918), each incorporated by reference.
[0055] In an aspect of the invention, the Cas9 endonuclease causes
a break one or more locations in foreign nucleic acid. These two
double strand breaks may cause a fragment of the genome to be
deleted. Even if repair pathways anneal the two ends, there will
still be a deletion in the genome. One or more deletions using the
mechanism will incapacitate the viral genome. The result is that
the transgenic organism will be free of viral infection.
[0056] In embodiments of the invention, nucleases cleave the genome
of a target virus. A nuclease is an enzyme capable of cleaving the
phosphodiester bonds between the nucleotide subunits of nucleic
acids. Endonucleases are enzymes that cleave the phosphodiester
bond within a polynucleotide chain. Some, such as Deoxyribonuclease
I, cut DNA relatively nonspecifically (without regard to sequence),
while many, typically called restriction endonucleases or
restriction enzymes, cleave only at very specific nucleotide
sequences. In a preferred embodiment of the invention, the Cas9
nuclease is incorporated into the compositions and methods of the
invention, however, it should be appreciated that any nuclease may
be utilized.
[0057] In preferred embodiments of the invention, the Cas9 nuclease
is used to cleave the genome. The Cas9 nuclease is capable of
creating a double strand break in the genome. The Cas9 nuclease has
two functional domains: RuvC and HNH, each cutting a different
strand. When both of these domains are active, the Cas9 causes
double strand breaks in the genome.
[0058] In some embodiments of the invention, insertions into the
genome can be designed to cause incapacitation, or altered genomic
expression. Additionally, insertions/deletions are also used to
introduce a premature stop codon either by creating one at the
double strand break or by shifting the reading frame to create one
downstream of the double strand break. Any of these outcomes of the
NHEJ repair pathway can be leveraged to disrupt the target gene.
The changes introduced by the use of the CRISPR/gRNA/Cas9 system
are permanent to the genome.
[0059] In some embodiments of the invention, at least one cut or
insertion is caused by the CRISPR/gRNA/Cas9 complex. In a preferred
embodiment, numerous insertions are caused in the genome, thereby
incapacitating the virus. In an aspect of the invention, the number
of insertions lowers the probability that the genome may be
repaired.
[0060] In some embodiments of the invention, at least one deletion
is caused by the CRISPR/gRNA/Cas9 complex. In a preferred
embodiment, numerous deletions are caused in the genome, thereby
incapacitating the virus. In an aspect of the invention, the number
of deletions lowers the probability that the genome may be
repaired. In a highly-preferred embodiment, the CRISPR/Cas9/gRNA
system of the invention causes significant genomic disruption,
resulting in effective destruction of the viral genome, while
leaving the host genome intact.
[0061] TALENs uses a nonspecific DNA-cleaving nuclease fused to a
DNA-binding domain that can be to target essentially any sequence.
For TALEN technology, target sites are identified and expression
vectors are made. Linearized expression vectors (e.g., by Notl) may
be used as template for mRNA synthesis. A commercially available
kit may be use such as the mMESSAGE mMACHINE SP6 transcription kit
from Life Technologies (Carlsbad, Calif.). See Joung & Sander,
2013, TALENs: a widely applicable technology for targeted genome
editing, Nat Rev Mol Cell Bio 14:49-55, incorporated by
reference.
[0062] TALENs and CRISPR methods provide one-to-one relationship to
the target sites, i.e. one unit of the tandem repeat in the TALE
domain recognizes one nucleotide in the target site, and the crRNA,
gRNA, or sgRNA of CRISPR/Cas system hybridizes to the complementary
sequence in the DNA target. Methods can include using a pair of
TALENs or a Cas9 protein with one gRNA to generate double-strand
breaks in the target. The breaks are then repaired via
non-homologous end-joining or homologous recombination (HR).
[0063] FIG. 5 shows ZFN being used to cut viral nucleic acid.
Briefly, the ZFN method includes introducing into the infected host
cell at least one vector (e.g., RNA molecule) encoding a targeted
ZFN 305 and, optionally, at least one accessory polynucleotide.
See, e.g., U.S. Pub. 2011/0023144 to Weinstein, incorporated by
reference The cell includes target sequence 311. The cell is
incubated to allow expression of the ZFN 305, wherein a
double-stranded break 317 is introduced into the targeted
chromosomal sequence 311 by the ZFN 305. In some embodiments, a
donor polynucleotide or exchange polynucleotide 321 is introduced.
Swapping a portion of the viral nucleic acid with irrelevant
sequence can fully interfere transcription or replication of the
viral nucleic acid. Target DNA 311 along with exchange
polynucleotide 321 may be repaired by an error-prone non-homologous
end-joining DNA repair process or a homology-directed DNA repair
process.
[0064] Typically, a ZFN comprises a DNA binding domain (i.e., zinc
finger) and a cleavage domain (i.e., nuclease) and this gene may be
introduced as mRNA (e.g., 5' capped, polyadenylated, or both). Zinc
finger binding domains may be engineered to recognize and bind to
any nucleic acid sequence of choice. See, e.g., Qu et al., 2013,
Zinc-finger-nucleases mediate specific and efficient excision of
HIV-1 proviral DAN from infected and latently infected human T
cells, Nucl Ac Res 41(16):7771-7782, incorporated by reference. An
engineered zinc finger binding domain may have a novel binding
specificity compared to a naturally-occurring zinc finger protein.
Engineering methods include, but are not limited to, rational
design and various types of selection. A zinc finger binding domain
may be designed to recognize a target DNA sequence via zinc finger
recognition regions (i.e., zinc fingers). See for example, U.S.
Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, incorporated by
reference. Exemplary methods of selecting a zinc finger recognition
region may include phage display and two-hybrid systems, and are
disclosed in U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S.
Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No.
6,410,248; U.S. Pat. No. 6,140,466; U.S. Pat. No. 6,200,759; and
U.S. Pat. No. 6,242,568, each of which is incorporated by
reference.
[0065] A ZFN also includes a cleavage domain. The cleavage domain
portion of the ZFNs may be obtained from any suitable endonuclease
or exonuclease such as restriction endonucleases and homing
endonucleases. See, for example, Belfort & Roberts, 1997,
Homing endonucleases: keeping the house in order, Nucleic Acids Res
25(17):3379-3388. A cleavage domain may be derived from an enzyme
that requires dimerization for cleavage activity. Two ZFNs may be
required for cleavage, as each nuclease comprises a monomer of the
active enzyme dimer. Alternatively, a single ZFN may comprise both
monomers to create an active enzyme dimer. Restriction
endonucleases present may be capable of sequence-specific binding
and cleavage of DNA at or near the site of binding. Certain
restriction enzymes (e.g., Type IIS) cleave DNA at sites removed
from the recognition site and have separable binding and cleavage
domains. For example, the Type IIS enzyme FokI, active as a dimer,
catalyzes double-stranded cleavage of DNA, at 9 nucleotides from
its recognition site on one strand and 13 nucleotides from its
recognition site on the other. The FokI enzyme used in a ZFN may be
considered a cleavage monomer. Thus, for targeted double-stranded
cleavage using a FokI cleavage domain, two ZFNs, each comprising a
FokI cleavage monomer, may be used to reconstitute an active enzyme
dimer. See Wah, et al., 1998, Structure of FokI has implications
for DNA cleavage, PNAS 95:10564-10569; U.S. Pat. No. 5,356,802;
U.S. Pat. No. 5,436,150; U.S. Pat. No. 5,487,994; U.S. Pub.
2005/0064474; U.S. Pub. 2006/0188987; and U.S. Pub. 2008/0131962,
each incorporated by reference.
[0066] In the ZFN-mediated process, a double stranded break
introduced into the target sequence by the ZFN is repaired, via
homologous recombination with the exchange polynucleotide, such
that the sequence in the exchange polynucleotide may be exchanged
with a portion of the target sequence. The presence of the double
stranded break facilitates homologous recombination and repair of
the break. The exchange polynucleotide may be physically integrated
or, alternatively, the exchange polynucleotide may be used as a
template for repair of the break, resulting in the exchange of the
sequence information in the exchange polynucleotide with the
sequence information in that portion of the target sequence. Thus,
a portion of the viral nucleic acid may be converted to the
sequence of the exchange polynucleotide. ZFN methods can include
using a vector to deliver a nucleic acid molecule encoding a ZFN
and, optionally, at least one exchange polynucleotide or at least
one donor polynucleotide to the infected cell.
[0067] Meganucleases are endodeoxyribonucleases characterized by a
large recognition site (double-stranded DNA sequences of 12 to 40
base pairs); as a result this site generally occurs only once in
any given genome. For example, the 18-base pair sequence recognized
by the I-SceI meganuclease would on average require a genome twenty
times the size of the human genome to be found once by chance
(although sequences with a single mismatch occur about three times
per human-sized genome). Meganucleases are therefore considered to
be the most specific naturally occurring restriction enzymes.
Meganucleases can be divided into five families based on sequence
and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and
PD-(D/E)XK. The most well studied family is that of the LAGLIDADG
proteins, which have been found in all kingdoms of life, generally
encoded within introns or inteins although freestanding members
also exist. The sequence motif, LAGLIDADG, represents an essential
element for enzymatic activity. Some proteins contained only one
such motif, while others contained two; in both cases the motifs
were followed by .about.75-200 amino acid residues having little to
no sequence similarity with other family members. Crystal
structures illustrates mode of sequence specificity and cleavage
mechanism for the LAGLIDADG family: (i) specificity contacts arise
from the burial of extended .beta.-strands into the major groove of
the DNA, with the DNA binding saddle having a pitch and contour
mimicking the helical twist of the DNA; (ii) full hydrogen bonding
potential between the protein and DNA is never fully realized;
(iii) cleavage to generate the characteristic 4-nt 3'-OH overhangs
occurs across the minor groove, wherein the scissile phosphate
bonds are brought closer to the protein catalytic core by a
distortion of the DNA in the central "4-base" region; (iv) cleavage
occurs via a proposed two-metal mechanism, sometimes involving a
unique "metal sharing" paradigm; (v) and finally, additional
affinity and/or specificity contacts can arise from "adapted"
scaffolds, in regions outside the core .alpha./.beta. fold. See
Silva et al., 2011, Meganucleases and other tools for targeted
genome engineering, Curr Gene Ther 11(1):11-27, incorporated by
reference.
[0068] Some embodiments of the invention may utilize modified
version of a nuclease. Modified versions of the Cas9 enzyme
containing a single inactive catalytic domain, either RuvC- or
HNH-, are called `nickases`. With only one active nuclease domain,
the Cas9 nickase cuts only one strand of the target DNA, creating a
single-strand break or `nick`. Similar to the inactive dCas9 (RuvC-
and HNH-), a Cas9 nickase is still able to bind DNA based on gRNA
specificity, though nickases will only cut one of the DNA strands.
The majority of CRISPR plasmids are derived from S. pyogenes and
the RuvC domain can be inactivated by a D10A mutation and the HNH
domain can be inactivated by an H840A mutation.
[0069] A single-strand break, or nick, is normally quickly repaired
through the HDR pathway, using the intact complementary DNA strand
as the template. However, two proximal, opposite strand nicks
introduced by a Cas9 nickase are treated as a double strand break,
in what is often referred to as a `double nick` or `dual nickase`
CRISPR system. A double-nick induced double strain break can be
repaired by either NHEJ or HDR depending on the desired effect on
the gene target. At these double strain breaks, insertions and
deletions are caused by the CRISPR/Cas9 complex. In an aspect of
the invention, a deletion is caused by positioning two double
strand breaks proximate to one another, thereby causing a fragment
of the genome to be deleted.
[0070] In some embodiments, a nuclease is a directed RNA nuclease
that cleaves RNA from viruses or viral transcripts. One targetable
RNA nuclease system is the Type III-A CRISPR-Cas Csm complex of
Thermus thermophilus (TtCsm). TtCsm is composed of five different
protein subunits (Csm1-Csm5) with an uneven stoichiometry and a
single crRNA of variable size (35-53 nt). The TtCsm crRNA content
is similar to the Type III-B Cmr complex, indicating that crRNAs
are shared among different subtypes. TtCsm cleaves complementary
target RNAs at multiple sites. Unlike Type I complexes,
interference by TtCsm does not proceed via initial base pairing by
a seed sequence. For discussion see Staals et al., 2014, RNA
Targeting by the type III-A CRISPR-Cas Csm complex of Thermus
thermophiles, Molecular Cell 56(4):518-530, incorporated by
reference. Thus aspects of the invention provide a non-human
transgenic organism comprising a transgene, wherein the transgene
comprises nucleic acid that encodes a targeting nuclease that can
be activated to digest foreign RNA. The nuclease may be TtCsm or
any other suitable targetable nuclease that cuts RNA.
[0071] In some embodiments, the invention includes the use of the
Dicer, the RNA-induced silencing complex (RISC), or both. Dicer,
also known as endoribonuclease Dicer or helicase with RNase motif,
is an enzyme of the RNase III family. Dicer cleaves double-stranded
RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded
RNA fragments called small interfering RNA and microRNA
respectively. These fragments are approximately 20-25 base pairs
long with a two-base overhang on the 3' end. Dicer facilitates the
activation of the RNA-induced silencing complex (RISC), which is
essential for RNA interference. RISC has a catalytic component
argonaute, which is an endonuclease capable of degrading messenger
RNA (mRNA).
[0072] RISC is a multi-protein complex, specifically a
ribonucleoprotein, which incorporates one strand of a
double-stranded RNA (dsRNA) fragment, such as small interfering RNA
(siRNA) or microRNA (miRNA). The single strand acts as a template
for RISC to recognize complementary messenger RNA (mRNA)
transcript. Once found, argonaute activates and cleaves the mRNA.
This process is called RNA interference (RNAi) and provides for
gene silencing and defense against viral infections.
[0073] The RNase III Dicer aids RISC in RNA interference by
cleaving dsRNA into 21-23 nucleotide long fragments with a
two-nucleotide 3' overhang. These dsRNA fragments are loaded into
RISC and each strand has a different fate based on the asymmetry
rule phenomenon.
[0074] The strand with the less stable 5' end is selected by the
argonaute and integrated into RISC. This strand is known as the
guide strand. The other strand, known as the passenger strand, is
degraded by RISC. RISC uses the bound guide strand to target
complementary 3'-untranslated regions (3'UTR) of mRNA transcripts
via Watson-Crick base pairing. RISC can now regulate gene
expression of the mRNA transcript in a number of ways. RISC
degrades target mRNA which reduces the levels of transcript
available to be translated by ribosomes. There are two main
requirements for mRNA degradation to take place: a near-perfect
complementary match between the guide strand and target mRNA
sequence; and a catalytically active argonaute protein, called a
`slicer`, to cleave the target mRNA. Also, RISC can modulate the
loading of ribosome and accessory factors in translation to repress
expression of the bound mRNA transcript. Translational repression
only requires a partial sequence match between the guide strand and
target mRNA. Translation can be regulated at the initiation step by
preventing the binding of the eukaryotic translation initiation
factor (eIF) to the 5' cap. It has been noted RISC can adeadenylate
the 3' poly(A) tail which might contribute to repression via the 5'
cap. RISC may also prevent the binding of the 60S ribosomal subunit
to the mRNA. Thus some aspects of the invention provide a non-human
transgenic organism comprising a transgene, wherein the transgene
comprises nucleic acid that encodes a targeting nuclease that can
be activated to digest foreign RNA.
[0075] Embodiments of the invention use a components of the
Dicer/RISC system that naturally occur in plants or provides for
the expression of an orthologous complex. In some embodiments, a
transgenic agricultural crop plant or livestock animal is provided
with a transgene for one or more component of the Dicer/RISC
system. During infection, an RNA-induced silencing complex (RISC)
is programmed with viral short-interfering RNAs (siRNAs) to target
the cognate viral RNA for degradation. A RISC complex gene may be
taken from Nicotiana benthamiana and cloned into the transgenic
organism. Discussion may be found in Ciomperlik et al., 2012, An
antiviral RISC isolated from Tobaccco rattle virus-infected plants,
Virology 412(1):117-124, incorporated by reference.
[0076] Argonaute proteins are a family of proteins that play a role
in RNA silencing as a component of the RNA-induced silencing
complex (RISC). The Argonaute of the archaeon Pyrococcus furiosus
(PfAgo) uses small 5'-phosphorylated DNA guides to cleave both
single stranded and double stranded DNA targets, and does not
utilize RNA as guide or target.
[0077] NgAgo uses 5' phosphorylated DNA guides (so called "gDNAs")
and appear to exhibit little preference for any certain guide
sequences and thus may offer a general-purpose DNA-guided
programmable nuclease. NgAgo does not require a PAM sequence, which
contributes to flexibility in choosing a genomic target. NgAgo also
appears to outperform Cas9 in GC-rich regions. NgAgo is only 887
amino acids in length. NgAgo randomly removes 1-20 nucleotides from
the cleavage site specified by the gDNA. Thus, PfAgo and NgAgo
represent potential DNA-guided programmable nucleases that may be
modified for use as a composition of the invention.
Targeting Sequence
[0078] The transgenic organism may express a targeting nuclease
that uses a targeting sequence such as a guide RNA (gRNA) to target
and digest foreign nucleic acid while avoiding off-target (e.g.,
self) digestion. The invention provides methods to avoid self
genome digestion. A targeting sequence may be pre-determined (e.g.,
to protect against a specific agricultural virus) and encoded
within the transgene.
[0079] FIG. 6 describes an exemplary method for selecting a gRNA
within the viral target region. A system or method of the invention
may be used to scan the viral coding sequence and finds the PAM for
the nuclease that is to be used. For example, where the digestion
system will include cas9, the system scan the target for NGG, where
N is any nucleotide. Upon finding the PAM in the viral genome, the
20 nucleotide string adjacent to the PAM within the viral genome
are read. This 20 nucleotide string is provisionally treated as a
potential sequence for the gRNA. Finally selecting the nucleotide
string for the gRNA involves determining if the nucleotide string
satisfies a similarity criteria for any region within the host
genome (i.e., a gRNA is only selected if there is no region within
the host genome that is similar according to a defined
criteria).
[0080] Any suitable similarity criteria may be used. For example,
one similarity criteria may be the requirement of a perfect match
for all 20 bases of the nucleotide string. Other criteria may
include that 19 bases match, or 18, etc. In a preferred embodiment,
the invention includes similarity criteria that balance the
requirement of actually finding a useful gRNA with the
probabilities of some matching portions in the host, i.e., the
possibility that even without a perfect 20 nt match, some of the
gRNA may still bind to the host genome and initiate nuclease
action. The includes similarity criteria that minimize off-target
action against the host genome.
[0081] FIG. 7 outlines a similarity criteria 601 according to
certain embodiments that may be automatically applied by, for
example, a computer system. To avoid digestion of host genome, the
system applies a search criteria that embodies certain principles.
The system preferably tries to avoid any target sequence with any
.gtoreq.12 nt DNA stretch homology to the human genome. When
homology to human genome is inevitable, the guide RNA candidate not
followed by PAM in the human genome would not lead to off-target
digestion, and should be given priority. If homologous sequences
and PAM both are present in the human genome, one should choose the
guide RNA candidate with low homology (e.g., <40% similar) to
human genome in the half next to PAM, where double strand break
happens.
[0082] To reach these principles, as diagrammed in FIG. 7, the
system reads in a 20 nt nucleotide string adjacent a PAM in the
viral sequence. The system examines the host genome for any segment
with .gtoreq.12 nt identity to the nucleotide string. If no such
segment is found (N), then that nucleotide string is provided as
the guide sequence to target that 20 nt in the viral genome. If
such a segment is found in the human genome (Y), then the system
determines if that segment in the host genome is adjacent to a PAM.
If that segment in the host genome is not adjacent to a PAM (N),
then that nucleotide string is provided as the guide sequence to
target that 20 nt in the viral genome. If that segment in the host
genome is adjacent to a PAM (Y), then the system determines if the
half of that segment that is closest to the PAM is less than 40%
similar to the nucleotide string. If the half of that segment that
is closest to the PAM is less than 40% similar to the nucleotide
string (Y), then that nucleotide string is provided as the guide
sequence to target that 20 nt in the viral genome. If the half of
that segment that is closest to the PAM is not less than 40%
similar to the nucleotide string, then the system reads in the next
20 nt nucleotide string in the viral genome sequence that is
adjacent to a PAM and repeats the steps on that next candidate
string. The cycle of steps is optionally repeated until at least
one guide sequence is provided. Optionally, the steps may be
repeated until several or all possible guide sequences are
provided.
[0083] In some embodiments of the invention, targeting sequences
are expressed within an organelle such as a chloroplast. Expression
within an organelle may be beneficial in protecting a gene, a
plasmid, plastid, a gene product, etc., from deleterious elements
such as endogenous plant RNAi pathways. In certain embodiments, a
gene is provided within a chloroplast or other organelle that
encodes nucleic acid that is complementary to a target gene or
locus a virus, parasite, or pest such as an insect. Preferably, the
gene is integrated into a genome of an organelle, such as the
chloroplast genome or a mitochondrial genome. The nucleic acid may
be expressed and used as a guide RNA, e.g., for a Cas9 enzyme
(which may also be present as a gene or protein in the organelle or
another organelle). It is also possible that the nucleic acid is a
dsRNA that triggers a lethal RNAi response in a pest. See e.g.,
Zhang, 2015, Full crop protection from an insect pest by expression
of long double-stranded RNAs in plastids, Science
347(6225):991-994, incorporated by reference. For example, an
organelle may have a gene (either integrated into its genome or
present as an independent particle such as a plasmid with its own
replication origin) that when transcribed into RNA is complementary
to a portion of a nucleic acid of a virus, parasite, or pest. The
transcribed RNA hybridizes to the portion that it is complementary
to and may trigger the RNAi system to destroy the target.
Expression
[0084] Methods of the invention may be used to create a transgenic
organism for agriculture that expresses a nuclease that digests
foreign nucleic acid thus protecting the organism from viral
infection. Any suitable expression pattern may be provided
including, for example, constitutive or conditional expression.
[0085] In some embodiments, the full nuclease is constitutively
expressed in all cells at all times. This may be beneficial for
providing a transgenic crop plant or livestock animal with a form
of immune system against viral infection. A CRISPR/Cas9 sequence
may be constitutively expressed and may respond to viral infection
by integrating fragments of viral nucleic acid into the clustered
repeats of the CRISPR. Those then may function as template for
guide RNAs during future infections.
[0086] In certain embodiments, the transgene or nuclease is only
expressed in certain cells such as the cells that the virus is
capable of infecting. An important characteristic of some viruses
is tropism. Tropism of a virus pertains to the types of cells,
tissues, and animal and plant species in which it can replicate. A
transgene can be under control of a tissue-specific promoter, for
example. Those include promoters controlling gene expression in a
tissue-dependent manner and according to the developmental stage of
the plant. The transgenes driven by these type of promoters will
only be expressed in tissues where the transgene product is
desired, leaving the rest of the tissues in the plant unmodified by
transgene expression. Tissue-specific promoters may be induced by
endogenous or exogenous factors, so they can be classified as
inducible promoters as well. Unlike constitutive expression of
genes, tissue-specific expression is the result of several
interacting levels of gene regulation. As such, it is then
preferable to use promoters from homologous or closely related
plant species to achieve efficient and reliable expression of
transgenes in particular tissues. Tissue promoters include
beta-amylase gene or barley hordein gene promoters (for seed gene
expression), tomato pz7 and pz130 gene promoters (for ovary gene
expression), tobacco RD2 gene promoter (for root gene expression),
banana TRX promoter and melon actin promoter (for fruit gene
expression) and others. Tissue specific promoters may include root
promoters such as those available from Pioneer Hi-Bred. Root
promoters enhance or suppress the expression of a linked gene in
root cells. Fruit promoters, such as those available from Calgene,
include fruit specific promoters that control the expression of
genes in mature ovary tissue of a fruit and in the receptacle
tissue of accessory fruits such as strawberry, apple and pear. Seed
promoters (e.g., available from Calgene) include transcription
cassettes having a seed-specific promoter and recombinant molecules
containing a seed-maturation promoter.
[0087] Additionally or alternatively, nuclease expression may be
dependent on an external event. For example, a transgene may be
under control of an inducible promoter linked to a small molecule.
Numerous inducible promoters are known in the art and include
chemically-regulated promoters such as those derived from organisms
such as yeast, E. coli, Drosophila or mammals. Inducible promoters
include alcohol-regulated promoters (e.g., available from
Syngenta). These provide a transcriptional system containing the
alcohol dehydrogenase I (alcA) gene promoter and the transactivator
protein AlcR. Different agricultural alcohol-based formulations are
used to control the expression of a gene of interest linked to the
alcA promoter. In some embodiments, an inducible promoter is a
tetracycline-regulated promoter, such as promoters available from
BASF AG. The tetracycline-responsive promoter systems can function
either to activate or repress gene expression system in the
presence of tetracycline. Some of the elements of the systems
include a tetracycline repressor protein (TetR), a tetracycline
operator sequence (tetO) and a tetracycline transactivator fusion
protein (tTA), which is the fusion of TetR and a herpes simplex
virus protein 16 (VP16) activation sequence. Inducible promoters
may include steroid-regulated promoters. Steroid-regulated
promoters suitable for use include those based on the rat
glucocorticoid receptor (GR), promoters based on the human estrogen
receptor (ER), promoters based on ecdysone receptors derived from
different moth species, as well as promoters from the
steroid/retinoid/thyroid receptor superfamily. In some embodiments,
an inducible promoter is metal-regulated. Promoters derived from
metallothionein (proteins that bind and sequester metal ions) genes
from yeast, mouse and human may be used to provide a promoter that
is regulated by metal. Additionally, inducible promoters may
include pathogenesis-related (PR) proteins that are induced in
plants in the presence of particular exogenous chemicals in
addition to being induced by pathogen infection. Salicylic acid,
ethylene and benzothiadiazole (BTH) are some of the inducers of PR
proteins. Such promoters have been derived from Arabidopsis and
maize PR genes.
[0088] Using an inducible promoter provides control over
conditional expression. For example, if a transgenic plant or
animal appears sick or is exposed to infection, it may be
administered a small molecule (e.g., via its feed) to initiate
expression of the nuclease. Or this could be done periodically as a
prophylactic measure. Thus in some aspects, the invention provides
non-human transgenic organism comprising a transgene, wherein the
transgene comprises nucleic acid that encodes a targeting nuclease
that can be activated to digest foreign nucleic acid, wherein the
transgene is under the control of a promoter. The promoter may be
an inducible promoter, for example, a chemically-regulated
promoters such as a tetracycline-regulated promoter. Additionally
or alternatively, the promoter may be a virus-dependent promoter,
so that nuclease expression is only turned on in infected
cells.
[0089] For two component nucleases (like CRISPR) one of the two
components may be expressed constitutively while the other is
expressed conditionally (e.g., under the control of an inducible
promoter). For example, the system may constitutively express CAS9
and have the guide RNA conditionally expressed (or vice-versa).
Cleave Foreign Nucleic Acid
[0090] As discussed above, methods of the invention may be used to
create a transgenic organism for agriculture that expresses a
nuclease that digests foreign nucleic acid thus protecting the
organism from viral infection. The nuclease may use a targeting
sequencing that can also be transgenically included in the
organism. Using the general methods described above, one may use
knowledge of specific viral genomes to design targeting sequences
against those viruses to so protect an organism.
[0091] FIG. 8 diagrams the avian flu virus genome for targets for
cleavage. The Avian flu virus genome has been described and
published. See, e.g., Pabbaraju et al., 2014, Full-genome analysis
of avian influenza A(H5N1) virus from a human, North America, 2013,
Emerg Inf Dis 20(5):887-891, incorporated by reference. One my
choose segments from that genome that meet the required similarity
criteria against the native genome of the transgenic organism and
include those segments as targeting sequences in a transgene. Thus,
where the invention is used to provide a transgenic poultry
organism, the animal can digest flu virus to avoid infection.
[0092] FIG. 9 shows a genome of a Bluetongue virus (BTV) for
targeting. Bluetongue Virus (BTV) is a pathogenic virus that causes
serious disease in livestock. The BTV genome has been described and
published. See Minakshi et al., 2012, Complete genome sequence of
Bluetongue virus serotype 16 of goat origin from India, J Virol
86(15):8337-8338, incorporated by reference. Using this information
and the similarity criteria one may create a transgenic animal such
as a sheep, cattle, or goat that itself digests Bluetongue virus
genetic material.
[0093] FIG. 10 diagrams the tobacco mosaic virus (TMV) genetic
material. TMV infects a wide range of plants, especially tobacco
and other members of the family Solanaceae. The TMV genome has been
described. See Goelet et al., 1982, Nucleotide sequence of tobacco
mosaic virus RNA, PNAS 79(19):5818-5822. A transgenic tobacco or
other plant of the family Solanaceae may be created wherein the
transgene encodes a targeting nuclease and a targeting sequence
specific to the TMV genome, allowing the plant to digest the
foreign nucleic acid.
[0094] FIG. 11 shows parts of the genome of banana bunchy top virus
(BBTV). BBTV is a major and significant disease that severely
effects banana crops. The BBTV genome has been described. See Burns
1995, The genome organization of banana bunchy top virus: analysis
of six ssDNA components, J Gen Vir 76:1471-1482, incorporated by
reference. Using this information and the similarity criteria and
methods described herein, methods of the invention may be used to
provide a transgenic banana that digests BBTV genetic material.
[0095] Using methods, organisms, or seeds as provided by the
invention, agriculture may be improved. For example, if livestock
show signs of infection, they may be fed a small molecule that
initiates transient expression of a nuclease to battle the
infection. The nuclease may be expressed in every cell or in a
tissue-specific manner. Additionally or alternatively, agricultural
organisms could be treated prophylatically to prevent any signs of
infection.
[0096] For additional background, see Yin et al., 2015, Multiplex
conditional mutagenesis using transgenic expression of Cas9 and
sgRNAs, Genetics 200:431-41; Xue et al., 2014, Efficient gene
knock-out and knock-in with transgenic Cas9 in Drosophila, G3
4:925-929; and Harrison et al., 2014, A CRISPR view of development,
Genes and Development 28:1859-1872, the contents of each of which
are incorporated by reference.
[0097] In the CRISPR-Cas system, short sequences (referred to as
"protospacers") from an invading viral genome are copied as
"spacers" between repetitive sequences in the CRISPR locus of the
host genome. The CRISPR locus is transcribed and processed into
short CRISPR RNAs (crRNAs) that guide the Cas to the complementary
genomic target sequence. There are at least eleven different
CRISPR-Cas systems, which have been grouped into three major types
(I-III). In the type I and II systems, nucleotides adjacent to the
protospacer in the targeted genome comprise the protospacer
adjacent motif (PAM). The PAM is essential for Cas to cleave its
target DNA, enabling the CRISPR-Cas system to differentiate between
the invading viral genome and the CRISPR locus in the host genome,
which does not incorporate the PAM. For additional details on this
fascinating prokaryotic adaptive immune response, see recent
reviews (Sorek et al. 2013; Terns and Terns 2014).
[0098] Transgenic expression of the gRNA has been demonstrated to
increase the frequency of targeted events. The expression of Cas9
can be restricted by placing it under the control of
tissue-specific regulatory sequences. Expression of Cas9 is
discussed in Xue, 2014, Efficient gene knock-out and knock-in with
transgenic Cas9 in Drosophila, G3 4(5):925-929 and in Yin, et al.,
2015, Multiplex conditional mutagenesis using transgenic expression
of Cas9 and sgRNAs, Genetics 200:431-441, both incorporated by
reference. Agriculturally important viral targets may include RNA
or ssDNA and Cas9 may be used to digest such nucleic acid. See
O'Connell et al., 2014, Programmable RNA recognition and cleavage
by CRISPR/Cas9, Nature 516:263-266; Price et al., Cas9-mediated
targeting of viral RNA in eukaryotic cells, PNAS 112(19):6164-6169;
Hwang et al., 2013, Heritable and precise zebrafish genome editing
using a CRISPR-Cas system, PLoSOne 8(7):e68708, each incorporated
by reference.
Incorporation by Reference
[0099] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes.
Equivalents
[0100] Various modifications of the invention and many further
embodiments thereof, in addition to those shown and described
herein, will become apparent to those skilled in the art from the
full contents of this document, including references to the
scientific and patent literature cited herein. The subject matter
herein contains important information, exemplification and guidance
that can be adapted to the practice of this invention in its
various embodiments and equivalents thereof.
EXAMPLES
Example 1
Digesting Viral Nucleic Acid I
[0101] Methods and materials of the present invention may be used
to digest foreign nucleic acid such as a genome of a hepatitis B
virus (HBV).
[0102] It may be preferable to receive annotations for the HBV
genome (i.e., that identify important features of the genome) and
choose a candidate for targeting by enzymatic degredation that lies
within one of those features, such as a viral replication origin, a
terminal repeat, a replication factor binding site, a promoter, a
coding sequence, and a repetitive region.
[0103] HBV, which is the prototype member of the family
Hepadnaviridae, is a 42 nm partially double stranded DNA virus,
composed of a 27 nm nucleocapsid core (HBcAg), surrounded by an
outer lipoprotein coat (also called envelope) containing the
surface antigen (HBsAg). The virus includes an enveloped virion
containing 3 to 3.3 kb of relaxed circular, partially duplex DNA
and virion-associated DNA-dependent polymerases that can repair the
gap in the virion DNA template and has reverse transcriptase
activities. HBV is a circular, partially double-stranded DNA virus
of approximately 3200 by with four overlapping ORFs encoding the
polymerase (P), core (C), surface (S) and X proteins. In infection,
viral nucleocapsids enter the cell and reach the nucleus, where the
viral genome is delivered. In the nucleus, second-strand DNA
synthesis is completed and the gaps in both strands are repaired to
yield a covalently closed circular DNA molecule that serves as a
template for transcription of four viral RNAs that are 3.5, 2.4,
2.1, and 0.7 kb long. These transcripts are polyadenylated and
transported to the cytoplasm, where they are translated into the
viral nucleocapsid and precore antigen (C, pre-C), polymerase (P),
envelope L (large), M (medium), S (small)), and transcriptional
transactivating proteins (X). The envelope proteins insert
themselves as integral membrane proteins into the lipid membrane of
the endoplasmic reticulum (ER). The 3.5 kb species, spanning the
entire genome and termed pregenomic RNA (pgRNA), is packaged
together with HBV polymerase and a protein kinase into core
particles where it serves as a template for reverse transcription
of negative-strand DNA. The RNA to DNA conversion takes place
inside the particles.
[0104] Numbering of basepairs on the HBV genome is based on the
cleavage site for the restriction enzyme EcoR1 or at homologous
sites, if the EcoR1 site is absent. However, other methods of
numbering are also used, based on the start codon of the core
protein or on the first base of the RNA pregenome. Every base pair
in the HBV genome is involved in encoding at least one of the HBV
protein. However, the genome also contains genetic elements which
regulate levels of transcription, determine the site of
polyadenylation, and even mark a specific transcript for
encapsidation into the nucleocapsid. The four ORFs lead to the
transcription and translation of seven different HBV proteins
through use of varying in-frame start codons. For example, the
small hepatitis B surface protein is generated when a ribosome
begins translation at the ATG at position 155 of the adw genome.
The middle hepatitis B surface protein is generated when a ribosome
begins at an upstream ATG at position 3211, resulting in the
addition of 55 amino acids onto the 5' end of the protein.
[0105] ORF P occupies the majority of the genome and encodes for
the hepatitis B polymerase protein. ORF S encodes the three surface
proteins. ORF C encodes both the hepatitis e and core protein. ORF
X encodes the hepatitis B X protein. The HBV genome contains many
important promoter and signal regions necessary for viral
replication to occur. The four ORFs transcription are controlled by
four promoter elements (preS1, preS2, core and X), and two enhancer
elements (Enh I and Enh II). All HBV transcripts share a common
adenylation signal located in the region spanning 1916-1921 in the
genome. Resulting transcripts range from 3.5 nucleotides to 0.9
nucleotides in length. Due to the location of the core/pregenomic
promoter, the polyadenylation site is differentially utilized. The
polyadenylation site is a hexanucleotide sequence (TATAAA) as
opposed to the canonical eukaryotic polyadenylation signal sequence
(AATAAA). The TATAAA is known to work inefficiently, suitable for
differential use by HBV.
[0106] There are four known genes encoded by the genome, called C,
X, P, and S. The core protein is coded for by gene C (HBcAg), and
its start codon is preceded by an upstream in-frame AUG start codon
from which the pre-core protein is produced. HBeAg is produced by
proteolytic processing of the pre-core protein. The DNA polymerase
is encoded by gene P. Gene S is the gene that codes for the surface
antigen (HBsAg). The HBsAg gene is one long open reading frame but
contains three in-frame start (ATG) codons that divide the gene
into three sections, pre-S1, pre-S2, and S. Because of the multiple
start codons, polypeptides of three different sizes called large,
middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) are produced.
The function of the protein coded for by gene X is not fully
understood but it is associated with the development of liver
cancer. It stimulates genes that promote cell growth and
inactivates growth regulating molecules.
[0107] HBV starts its infection cycle by binding to the host cells
with PreS1. Guide RNA against PreS1 locates at the 5' end of the
coding sequence. Endonuclease digestion will introduce
insertion/deletion, which leads to frame shift of PreS1
translation. HBV replicates its genome through the form of long
RNA, with identical repeats DR1 and DR2 at both ends, and RNA
encapsidation signal epsilon at the 5' end. The reverse
transcriptase domain (RT) of the polymerase gene converts the RNA
into DNA. Hbx protein is a key regulator of viral replication, as
well as host cell functions. Digestion guided by RNA against RT
will introduce insertion/deletion, which leads to frame shift of RT
translation. Guide RNAs sgHbx and sgCore can not only lead to frame
shift in the coding of Hbx and HBV core protein, but also deletion
the whole region containing DR2-DR1-Epsilon. The four sgRNA in
combination can also lead to systemic destruction of HBV genome
into small pieces.
[0108] HBV replicates its genome by reverse transcription of an RNA
intermediate. The RNA templates is first converted into
single-stranded DNA species (minus-strand DNA), which is
subsequently used as templates for plus-strand DNA synthesis. DNA
synthesis in HBV use RNA primers for plus-strand DNA synthesis,
which predominantly initiate at internal locations on the
single-stranded DNA. The primer is generated via an RNase H
cleavage that is a sequence independent measurement from the 5' end
of the RNA template. This 18 nt RNA primer is annealed to the 3'
end of the minus-strand DNA with the 3' end of the primer located
within the 12 nt direct repeat, DR1. The majority of plus-strand
DNA synthesis initiates from the 12 nt direct repeat, DR2, located
near the other end of the minus-strand DNA as a result of primer
translocation. The site of plus-strand priming has consequences. In
situ priming results in a duplex linear (DL) DNA genome, whereas
priming from DR2 can lead to the synthesis of a relaxed circular
(RC) DNA genome following completion of a second template switch
termed circularization. It remains unclear why hepadnaviruses have
this added complexity for priming plus-strand DNA synthesis, but
the mechanism of primer translocation is a potential therapeutic
target. As viral replication is necessary for maintenance of the
hepadnavirus (including the human pathogen, hepatitis B virus)
chronic carrier state, understanding replication and uncovering
therapeutic targets is critical for limiting disease in
carriers.
[0109] In some embodiments, systems and methods of the invention
target the HBV genome by finding a nucleotide string within a
feature such as PreS1.
[0110] Guide RNA against PreS1 locates at the 5' end of the coding
sequence. Thus it is a good candidate for targeting because it
represents one of the 5'-most targets in the coding sequence.
Endonuclease digestion will introduce insertion/deletion, which
leads to frame shift of PreS1 translation. HBV replicates its
genome through the form of long RNA, with identical repeats DR1 and
DR2 at both ends, and RNA encapsidation signal epsilon at the 5'
end.
[0111] The reverse transcriptase domain (RT) of the polymerase gene
converts the RNA into DNA. Hbx protein is a key regulator of viral
replication, as well as host cell functions. Digestion guided by
RNA against RT will introduce insertion/deletion, which leads to
frame shift of RT translation.
[0112] Guide RNAs sgHbx and sgCore can not only lead to frame shift
in the coding of Hbx and HBV core protein, but also deletion the
whole region containing DR2-DR1-Epsilon. The four sgRNA in
combination can also lead to systemic destruction of HBV genome
into small pieces. In some embodiments, method of the invention
include creating one or several guide RNAs against key features
within a genome such as the HBV genome. To achieve the CRISPR
activity in cells, expression plasmids coding cas9 and guide RNAs
are delivered to cells of interest (e.g., cells carrying HBV DNA).
To demonstrate in an in vitro assay, anti-HBV effect may be
evaluated by monitoring cell proliferation, growth, and morphology
as well as analyzing DNA integrity and HBV DNA load in the
cells.
[0113] The described method may be validated using an in vitro
assay. To demonstrate, an in vitro assay is performed with cas9
protein and DNA amplicons flanking the target regions. Here, the
target is amplified and the amplicons are incubated with cas9 and a
gRNA having the selected nucleotide sequence for targeting. As
shown in FIG. 12, DNA electrophoresis shows strong digestion at the
target sites.
[0114] FIG. 12 shows a gel resulting from an in vitro CRISPR assay
against HBV. Lanes 1, 3, and 6: PCR amplicons of HBV genome
flanking RT, Hbx-Core, and PreS1. Lane 2, 4, 5, and 7: PCR
amplicons treated with sgHBV-RT, sgHBV-Hbx, sgHBV-Core,
sgHBV-PreS1. The presence of multiple fragments especially visible
in lanes 5 and 7 show that sgHBV-Core and sgHBV-PreS1 provide
especially attractive targets in the context of HBV and that use of
systems and methods of the invention may be shown to be effective
by an in vitro validation assay.
Example 2
Digesting Viral Nucleic Acid II
[0115] An exemplary assay shows the digestion of viral nucleic
acid.
[0116] Burkitt's lymphoma cell lines Raji, Namalwa, and DG-75 were
obtained from ATCC and cultured in RPMI 1640 supplemented with 10%
FBS and PSA, following ATCC recommendation. Human primary lung
fibroblast IMR-90 was obtained from Coriell and cultured in
Advanced DMEM/F-12 supplemented with 10% FBS and PSA.
[0117] Plasmids consisting of a U6 promoter driven chimeric guide
RNA (sgRNA) and a ubiquitous promoter driven Cas9 were obtained
from addgene, as described by Cong L et al. (2013) Multiplex Genome
Engineering Using CRISPR/Cas Systems. Science 339:819-823.
[0118] FIG. 13 shows a plasmid according to certain embodiments. An
EGFP marker fused after the Cas9 protein allowed selection of
Cas9-positive cells. A modified chimeric guide RNA stem-loop design
was adapted for more efficient Pol-III transcription and more
stable stem-loop structure (Chen B et al. (2013) Dynamic Imaging of
Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas
System. Cell 155:1479-1491).
[0119] We obtained pX458 from Addgene, Inc. A modified CMV promoter
with a synthetic intron (pmax) was PCR amplified from Lonza control
plasmid pmax-GFP. A modified guide RNA sgRNA(F+E) was ordered from
IDT. EBV replication origin oriP was PCR amplified from B95-8
transformed lymphoblastoid cell line GM12891. We used standard
cloning protocols to clone pmax, sgRNA(F+E) and oriP to pX458, to
replace the original CAG promoter, sgRNA and f1 origin. We designed
EBV sgRNA based on the B95-8 reference, and ordered DNA oligos from
IDT. The original sgRNA place holder in pX458 serves as the
negative control.
[0120] Lymphocytes are known for being resistant to lipofection,
and therefore we used nucleofection for DNA delivery into Raji
cells. We chose the Lonza pmax promoter to drive Cas9 expression as
it offered strong expression within Raji cells. We used the Lonza
Nucleofector II for DNA delivery. 5 million Raji or DG-75 cells
were transfected with 5 ug plasmids in each 100-ul reaction. Cell
line Kit V and program M-013 were used following Lonza
recommendation. For IMR-90, 1 million cells were transfected with 5
ug plasmids in 100 ul Solution V, with program T-030 or X-005. 24
hours after nucleofection, we observed obvious EGFP signals from a
small proportion of cells through fluorescent microscopy. The
EGFP-positive cell population decreased dramatically after that,
however, and we measured <10% transfection efficiency 48 hours
after nucleofection. We attributed this transfection efficiency
decrease to the plasmid dilution with cell division. To actively
maintain the plasmid level within the host cells, we redesigned the
CRISPR plasmid to include the EBV origin of replication sequence,
oriP. With active plasmid replication inside the cells, the
transfection efficiency rose to >60%.
[0121] To design guide RNA targeting the EBV genome, we relied on
the EBV reference genome from strain B95-8.
[0122] FIG. 14 diagrams the EBV genome. We targeted six regions
with seven guide RNA designs for different genome editing purposes.
The guide RNAs are listed in Table S1 in Wang and Quake, 2014,
RNA-guided endonuclease provides a therapeutic strategy to cure
latent herpesviridae infection, PNAS 111(36):13157-13162 and in the
Supporting Information to that article published online at the PNAS
website, and the contents of both of those documents are
incorporated by reference for all purposes.
[0123] EBNA1 is crucial for many EBV functions including gene
regulation and latent genome replication. We targeted guide RNA
sgEBV4 and sgEBV5 to both ends of the EBNA1 coding region in order
to excise this whole region of the genome. Guide RNAs sgEBV1, 2 and
6 fall in repeat regions, so that the success rate of at least one
CRISPR cut is multiplied. These "structural" targets enable
systematic digestion of the EBV genome into smaller pieces. EBNA3C
and LMP1 are essential for host cell transformation, and we
designed guide RNAs sgEBV3 and sgEBV7 to target the 5' exons of
these two proteins respectively.
EBV Genome Editing
[0124] The double-strand DNA breaks generated by CRISPR are
repaired with small deletions. These deletions will disrupt the
protein coding and hence create knockout effects. SURVEYOR assays
confirmed efficient editing of individual sites. Beyond the
independent small deletions induced by each guide RNA, large
deletions between targeting sites can systematically destroy the
EBV genome.
[0125] FIG. 15 shows genomic context around guide RNA sgEBV2 and
PCR primer locations.
[0126] FIG. 16 shows a large deletion induced by sgEBV2, where lane
1-3 are before, 5 days after, and 7 days after sgEBV2 treatment,
respectively. Guide RNA sgEBV2 targets a region with twelve 125-bp
repeat units (FIG. 8). PCR amplicon of the whole repeat region gave
a .about.1.8-kb band (FIG. 16). After 5 or 7 days of sgEBV2
transfection, we obtained .about.0.4-kb bands from the same PCR
amplification (FIG. 16). The .about.1.4-kb deletion is the expected
product of repair ligation between cuts in the first and the last
repeat unit (FIG. 15).
[0127] DNA sequences flanking sgRNA targets were PCR amplified with
Phusion DNA polymerase. SURVEYOR assays were performed following
manufacturer's instruction. DNA amplicons with large deletions were
TOPO cloned and single colonies were used for Sanger sequencing.
EBV load was measured with Taqman digital PCR on Fluidigm BioMark.
A Taqman assay targeting a conserved human locus was used for human
DNA normalization. 1 ng of single-cell whole-genome amplification
products from Fluidigm C1 were used for EBV quantitative PCR. We
further demonstrated that it is possible to delete regions between
unique targets (FIG. 10). Six days after sgEBV4-5 transfection, PCR
amplification of the whole flanking region (with primers EBV4F and
5R) returned a shorter amplicon, together with a much fainter band
of the expected 2 kb (FIG. 16).
[0128] FIG. 17 shows that Sanger sequencing of amplicon clones
confirmed the direct connection of the two expected cutting sites.
A similar experiment with sgEBV3-5 also returned an even larger
deletion, from EBNA3C to EBNA1. Additional information such as
primer design is shown in Wang and Quake, 2014, RNA-guided
endonuclease provides a therapeutic strategy to cure latent
herpesviridae infection, PNAS 111(36):13157-13162 and in the
Supporting Information to that article published online at the PNAS
website, and the contents of both of those documents are
incorporated by reference for all purposes.
[0129] Essential Targets For EBV Treatment. The seven guide RNAs in
our CRISPR cocktail target three different categories of sequences
which are important for EBV genome structure, host cell
transformation, and infection latency, respectively. To understand
the most essential targets for effective EBV treatment, we
transfected Raji cells with subsets of guide RNAs. Although
sgEBV4/5 reduced the EBV genome by 85%, they could not suppress
cell proliferation as effectively as the full cocktail. Guide RNAs
targeting the structural sequences (sgEBV1/2/6) could stop cell
proliferation completely, despite not eliminating the full EBV load
(26% decrease). We conclude that systematic destruction of EBV
genome structure appears to be more effective than targeting
specific key proteins for EBV treatment.
* * * * *